There has been a growing interest in the manufacture and use of microfluidic systems for the acquisition of chemical and biochemical information. Techniques commonly associated with the semiconductor electronics industry such as photolithography, wet chemical etching, etc., are being used in the fabrication of these microfluidic systems. The term “microfluidic”, refers to a system or device or “chip” having channels and chambers which are generally fabricated at the micron or submicron scale, e.g., having at least once cross-sectional dimension in the range of from about 0.1 μm to about 500 μm. Early discussion of the use of planar chip technology for the fabrication of microfluidic systems is provided in Manz et al., Trends in Anal. Chem. (1990) 10(5):144–149 and Manz et al., Adv. in Chromatog. (1993) 33:1–66, which describe the fabrication of such fluidic devices, and particularly microcapillary devices, in silicon and glass substrates.
Applications of microfluidic systems are myriad. For example, International Patent Appln. WO 96/04547 describes the use of microfluidic systems for capillary electrophoresis, liquid chromatography, flow injection analysis, and chemical reaction and synthesis. U.S. Pat. No. 5,942,443 entitled “HIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROFLUIDIC DEVICES”, issued on Aug. 24, 1999 discloses wide ranging applications of microfluidic systems in rapidly assaying large numbers of compounds for their effects on chemical, and preferably biochemical systems. Biochemical systems include the full range of catabolic and anabolic reactions which occur in living systems including enzymatic, binding, signaling and other reactions. Biochemical systems of particular interest include receptor-ligand interactions, enzyme-substrate interactions, cellular signaling pathways, transport reactions involving model barrier systems (e.g., cells or membrane fractions) for bio-availability screening, and a variety of other general systems.
One of the major advances in recent times has been the adaptation of microfluidic devices to the performance of the polymerase chain reaction (PCR) and other cyclic polymerase-mediated reactions. However, a significant problem faced by experimenters has been the control of process parameters such as temperature, reagent concentration, buffers, salts, other materials, and the like. In particular, PCR should be carried out at precisely controlled temperatures. For example, PCR is typically based on three discrete, multiply repeated steps: denaturation of a DNA template, annealing of a primer to the denatured DNA template, and extension of the primer with a polymerase to create a nucleic acid complementary to the template. The conditions under which these steps are performed are well established in the art. Each step has distinct temperature and time requirements typically as follows:
Denaturation96° C., 15 secondsPrimer Annealing55° C., 30 secondsPrimer Extension72° C., 1.5 minutes
See Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.;1990). Generally, microfluidic systems are well suited to the performance of PCR because they allow rapid temperature changes, quickly providing the correct temperature at each step. Further, because the microfluidic elements are extremely small in comparison to the mass of the substrate in which they are fabricated, the heat can be highly localized, e.g., it dissipates into and from the substrate before it affects other fluidic elements within the device.
In addition to efficient temperature control, an experimenter attempting to run PCR must overcome a second problem. Often the efficiency of amplification reactions is compromised by primer self-annealing (“primer dimer”) as well as larger non specific side-reaction products arising due to inefficient reaction conditions. Such nonspecific fragments adversely affect the yield of desired specific fragments through competition with the specific target in the reaction. Furthermore, the nonspecific fragments that are approximately the same size as the specific product can cause erroneous interpretation of results. Researchers have concluded that these nonspecific side reaction products originate from DNA polymerase catalyzed extension of partially annealed 3′ ends of primers to nonspecific sites in complex DNA under ambient temperature conditions. Therefore, it appears that efficiencies of thermostable DNA polymerases are greatly reduced at ambient temperature relative to their peak efficiencies at higher temperatures.
A “Hot Start” PCR method was developed as a means of reducing the amplification of nonspecific products. See, e.g., D'Aquila et al., (1991) Nucleic Acids Res. 19:37–49. In the earlier methods, one of the reaction components was withheld from the reaction until the reaction mixture was heated to a temperature greater than the annealing temperature, followed by the addition of the missing component. This approach causes the partially annealed 3′ primer ends to melt away from nonspecific sites, before they can be extended. Therefore, Hot Start PCR improves product yield and specificity. More recent approaches to “Hot Start” PCR include the use of a heat-labile wax or jelly barrier that melts and permits mixing of aqueous components at an elevated temperature. Chou et al., (1992) Nucleic Acids Res, 20:1717–1723. A third method utilizes a monoclonal antibody for deactivating Taq DNA Polymerase at ambient temperature. When the reaction mixture is heated to the denaturation temperature, the deactivation of the polymerase is reversed thereby facilitating amplification of specific targets. Kellogg et al., (1994) Biotechniques 16: 1134–1137. Although all of the above described methods are a significant improvement over simple PCR, a common problem associated with all these methods is that these methods are cumbersome to use and time consuming when working with multiple samples.
For the foregoing reasons, there is a need for efficient methods, devices and systems for performing temperature dependent reactions, such as hot start PCR, on multiple sample targets. The present invention satisfies this and other needs.